Abstract
Eukaryotic gene expression is dependent on the modification of the first transcribed nucleotide of pre-mRNA by the addition of the 7-methylguanosine cap. The cap protects transcripts from exonucleases and recruits complexes which mediate transcription elongation, processing and translation initiation. The cap is synthesized by a series of reactions which link 7-methylguanosine to the first transcribed nucleotide via a 5' to 5' triphosphate bridge. In mammals, cap synthesis is catalysed by the sequential action of RNGTT (RNA guanylyltransferase and 5'-phosphatase) and RNMT (RNA guanine-7 methyltransferase), enzymes recruited to RNA pol II (polymerase II) during the early stages of transcription. We recently discovered that the mammalian cap methyltransferase is a heterodimer consisting of RNMT and the RNMT-activating subunit RAM (RNMT-activating mini-protein). RAM activates and stabilizes RNMT and thus is critical for cellular cap methylation and cell viability. In the present study we report that RNMT interacts with the N-terminal 45 amino acids of RAM, a domain necessary and sufficient for maximal RNMT activation. In contrast, smaller components of this RAM domain are sufficient to stabilize RNMT. RAM functions in the nucleus and we report that nuclear import of RAM is dependent on PY nuclear localization signals and Kapβ2 (karyopherin β2) nuclear transport protein.
Publication types
-
Research Support, Non-U.S. Gov't
MeSH terms
-
Cell Line
-
Cell Nucleus / enzymology
-
Cell Nucleus / metabolism*
-
Glutathione Transferase / genetics
-
Glutathione Transferase / metabolism
-
Humans
-
Methylation
-
Methyltransferases / antagonists & inhibitors
-
Methyltransferases / genetics
-
Methyltransferases / metabolism*
-
Mutant Proteins / antagonists & inhibitors
-
Mutant Proteins / chemistry
-
Mutant Proteins / metabolism
-
Nuclear Localization Signals / antagonists & inhibitors
-
Nuclear Localization Signals / chemistry
-
Nuclear Localization Signals / genetics
-
Nuclear Localization Signals / metabolism*
-
Protein Interaction Domains and Motifs
-
Protein Stability
-
Protein Transport
-
RNA Caps / antagonists & inhibitors
-
RNA Caps / metabolism
-
RNA Precursors / antagonists & inhibitors
-
RNA Precursors / metabolism
-
RNA Stability
-
RNA, Messenger / antagonists & inhibitors
-
RNA, Messenger / metabolism
-
RNA, Small Interfering
-
RNA-Binding Proteins / antagonists & inhibitors
-
RNA-Binding Proteins / chemistry
-
RNA-Binding Proteins / genetics
-
RNA-Binding Proteins / metabolism*
-
Recombinant Fusion Proteins / chemistry
-
Recombinant Fusion Proteins / metabolism
-
Recombinant Proteins / chemistry
-
Recombinant Proteins / metabolism
-
beta Karyopherins / antagonists & inhibitors
-
beta Karyopherins / genetics
-
beta Karyopherins / metabolism*
Substances
-
Mutant Proteins
-
Nuclear Localization Signals
-
RNA Caps
-
RNA Precursors
-
RNA, Messenger
-
RNA, Small Interfering
-
RNA-Binding Proteins
-
Recombinant Fusion Proteins
-
Recombinant Proteins
-
TNPO1 protein, human
-
TNPO2 protein, human
-
beta Karyopherins
-
Methyltransferases
-
RAMAC protein, human
-
mRNA (guanine(N7))-methyltransferase
-
Glutathione Transferase